K562 cells are used as a model to study the control of human globin gene expression. They express embryonic and fetal but not adult globin genes. Results suggest that the lack of transcription of the adult beta-globin gene results from an alteration of a regulating trans-acting factors(s). Our objectives are to provide evidence for the existence of such a factor, to elucidate its nature as an activator or a repressor and to set up an in vitro assay for its measurement. Globin promoters activity is deduced from the transient expression of the bacterial CAT enzyme in K562 cells transfected with plasmids in which transcription of the CAT gene is driven by various globin gene promoters. In competition experiments, cells have been cotransfected with these plasmids and an excess of the corresponding globin promoter. However, low efficiency and poor reproducibility of transfection impaired analysis of the results. Therefore, details of the transfection procedure have been thoroughly worked out to optimize reproducibility and efficiency. Since competition experiments required final DNA concentrations found to be inhibitory of CAT expression in control experiments, we are now constructing competitor plasmids containing multiple copies of the globin promoter sequences. Experiments conducted with K562 cells grown in the presence of 5-azacytidine, did not provide evidence for expression of the CAT plasmid containing the beta-promoter, but showed non-specific enhancement of expression of the other CAT plasmids. Finally, we are attempting to develop an assay for trans-acting factors that would allow use to track them in a purification scheme. We constructed SP6 promoter-containing plasmids in which the globin promoters are used as templates for in vitro transcription. We hope to be able to set up conditions in which binding of proteins to the promoter will result in premature transcription termination. Labelled transcripts could then be analyzed by polyacrylamide gel electrophoresis and autoradiography.